Differential Effects of Reperfusion on Cardiac Mitochondrial Subpopulations in a Preclinical Porcine Model of Acute Myocardial Infarction.

Acute myocardial infarction (AMI) leads to localized cardiac ischemia and can be fatal if untreated. Despite being treatable, the threat of ischemia-reperfusion (IR) injury remains high. Mitochondria are central to both propagation and mitigation of IR injury, and cardiac mitochondria are categorized into two major subtypes-subsarcolemmal and interfibrillar mitochondria (SSM and IFM, respectively). We hypothesized that, in our pre-clinical porcine model of AMI, SSM and IFM are differentially affected by reperfusion. AMI was induced in female pigs by balloon occlusion of the left anterior descending artery for 45 min, followed by 4 h of reperfusion. At the end of reperfusion, animals were euthanized. Cardiac SSM and IFM from the affected ischemic area and a nearby non-ischemic area were isolated to compare mitochondrial function using substrates targeting mitochondrial electron transport chain complexes I and II. Despite detecting overall significant differences in mitochondrial function including yield, mitochondrial S3 and S4 respirations, and calcium retention, consistent individual functional differences in the two mitochondrial subpopulations were not observed, both between the two mitochondrial subtypes, as well as between the ischemic and non-ischemic tissue. Nonetheless, this study describes the mitochondrial subtype response within the initial few hours of reperfusion in a clinically relevant model of AMI, which provides valuable information needed to develop novel mitochondrially targeted therapies for AMI.

Sodium Nitroprusside-Enhanced Cardiopulmonary Resuscitation Improves Blood Flow by Pulmonary Vasodilation Leading to Higher Oxygen Requirements.

Sodium nitroprusside-enhanced cardiopulmonary resuscitation has shown superior resuscitation rates and neurologic outcomes in large animal models supporting the need for a randomized human clinical trial. This study is the first to show nonselective pulmonary vasodilation as a potential mechanism for the hemodynamic benefits. The pulmonary shunting that is created requires increased oxygen treatment, but the overall improvement in blood flow increases minute oxygen delivery to tissues. In this context, hypoxemia is an important safety endpoint and a 100% oxygen ventilation strategy may be necessary for the first human clinical trial.

MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload.

Heart failure remains a major public-health problem with an increase in the number of patients worsening from this disease. Despite current medical therapy, the condition still has a poor prognosis. Heart failure is complex but mitochondrial dysfunction seems to be an important target to improve cardiac function directly. Our goal was to analyze the effects of MitoQ (100 µM in drinking water) on the development and progression of heart failure induced by pressure overload after 14 weeks. The main findings are that pressure overload-induced heart failure in rats decreased cardiac function in vivo that was not altered by MitoQ. However, we observed a reduction in right ventricular hypertrophy and lung congestion in heart failure animals treated with MitoQ. Heart failure also decreased total mitochondrial protein content, mitochondrial membrane potential in the intermyofibrillar mitochondria. MitoQ restored membrane potential in IFM but did not restore mitochondrial protein content. These alterations are associated with the impairment of basal and stimulated mitochondrial respiration in IFM and SSM induced by heart failure. Moreover, MitoQ restored mitochondrial respiration in heart failure induced by pressure overload. We also detected higher levels of hydrogen peroxide production in heart failure and MitoQ restored the increase in ROS production. MitoQ was also able to improve mitochondrial calcium retention capacity, mainly in the SSM whereas in the IFM we observed a small alteration. In summary, MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload, by decreasing hydrogen peroxide formation, improving mitochondrial respiration and improving mPTP opening.

Role of epinephrine and extracorporeal membrane oxygenation in the management of ischemic refractory ventricular fibrillation: a randomized trial in pigs.

xtracorporeal membrane oxygenation (ECMO) is used in cardiopulmonary resuscitation (CPR) of refractory cardiac arrest. We used a 2×2 study design to compare ECMO versus CPR and epinephrine versus placebo in a porcine model of ischemic refractory ventricular fibrillation (VF). Pigs underwent 5 minutes of untreated VF, 10 minutes of CPR, and were randomized to receive epinephrine versus placebo for another 35 minutes. Animals were further randomized to LAD reperfusion at minute 45 with ongoing CPR versus veno-arterial ECMO cannulation at minute 45 of CPR and subsequent LAD reperfusion. Four-hour survival was improved with ECMO while epinephrine showed no effect.

Early Effects of Prolonged Cardiac Arrest and Ischemic Postconditioning during Cardiopulmonary Resuscitation on Cardiac and Brain Mitochondrial Function in Pigs.

BACKGROUND: Out-of-hospital cardiac arrest (CA) is a prevalent medical crisis resulting in severe injury to the heart and brain and an overall survival of less than 10%. Mitochondrial dysfunction is predicted to be a key determinant of poor outcomes following prolonged CA. However, the onset and severity of mitochondrial dysfunction during CA and cardiopulmonary resuscitation (CPR) is not fully understood. Ischemic postconditioning (IPC), controlled pauses during the initiation of CPR, has been shown to improve cardiac function and neurologically favorable outcomes after 15min of CA. We tested the hypothesis that mitochondrial dysfunction develops during prolonged CA and can be rescued with IPC during CPR (IPC-CPR). METHODS: A total of 63 swine were randomized to no ischemia (Naïve), 19min of ventricular fibrillation (VF) CA without CPR (Untreated VF), or 15min of CA with 4min of reperfusion with either standard CPR (S-CPR) or IPC-CPR. Mitochondria were isolated from the heart and brain to quantify respiration, rate of ATP synthesis, and calcium retention capacity (CRC). Reactive oxygen species (ROS) production was quantified from fresh frozen heart and brain tissue. RESULTS: Compared to Naïve, Untreated VF induced cardiac and brain ROS overproduction concurrent with decreased mitochondrial respiratory coupling and CRC, as well as decreased cardiac ATP synthesis. Compared to Untreated VF, S-CPR attenuated brain ROS overproduction but had no other effect on mitochondrial function in the heart or brain. Compared to Untreated VF, IPC-CPR improved cardiac mitochondrial respiratory coupling and rate of ATP synthesis, and decreased ROS overproduction in the heart and brain. CONCLUSIONS: Fifteen minutes of VF CA results in diminished mitochondrial respiration, ATP synthesis, CRC, and increased ROS production in the heart and brain. IPC-CPR attenuates cardiac mitochondrial dysfunction caused by prolonged VF CA after only 4min of reperfusion, suggesting that IPC-CPR is an effective intervention to reduce cardiac injury. However, reperfusion with both CPR methods had limited effect on mitochondrial function in the brain, emphasizing an important physiological divergence in post-arrest recovery between those two vital organs.

Sodium nitroprusside enhanced cardiopulmonary resuscitation improves short term survival in a porcine model of ischemic refractory ventricular fibrillation.

INTRODUCTION: Sodium nitroprusside (SNP) enhanced CPR (SNPeCPR) demonstrates increased vital organ blood flow and survival in multiple porcine models. We developed a new, coronary occlusion/ischemia model of prolonged resuscitation, mimicking the majority of out-of-hospital cardiac arrests presenting with shockable rhythms. HYPOTHESIS: SNPeCPR will increase short term (4-h) survival compared to standard 2015 Advanced Cardiac Life Support (ACLS) guidelines in an ischemic refractory ventricular fibrillation (VF), prolonged CPR model. METHODS: Sixteen anesthetized pigs had the ostial left anterior descending artery occluded leading to ischemic VF arrest. VF was untreated for 5min. Basic life support was performed for 10min. At minute 10 (EMS arrival), animals received either SNPeCPR (n=8) or standard ACLS (n=8). Defibrillation (200J) occurred every 3min. CPR continued for a total of 45min, then the balloon was deflated simulating revascularization. CPR continued until return of spontaneous circulation (ROSC) or a total of 60min, if unsuccessful. SNPeCPR animals received 2mg of SNP at minute 10 followed by 1mg every 5min until ROSC. Standard ACLS animals received 0.5mg epinephrine every 5min until ROSC. Primary endpoints were ROSC and 4-h survival. RESULTS: All SNPeCPR animals (8/8) achieved sustained ROSC versus 2/8 standard ACLS animals within one hour of resuscitation (p=0.04). The 4-h survival was significantly improved with SNPeCPR compared to standard ACLS, 7/8 versus 1/8 respectively, p=0.0019. CONCLUSION: SNPeCPR significantly improved ROSC and 4-h survival compared with standard ACLS CPR in a porcine model of prolonged ischemic, refractory VF cardiac arrest.

Intracoronary Poloxamer 188 Prevents Reperfusion Injury in a Porcine Model of ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Poloxamer 188 (P188) is a nonionic triblock copolymer believed to prevent cellular injury after ischemia and reperfusion. OBJECTIVES: This study compared intracoronary infusion of P188 immediately after reperfusion with delayed infusion through a peripheral intravenous catheter in a porcine model of ST segment elevation myocardial infarction (STEMI). Cellular and mitochondrial injury were assessed. METHODS: STEMI was induced in 55 pigs using 45 minutes of endovascular coronary artery occlusion. Pigs were then randomized to four groups: control, immediate intracoronary (IC) P188, delayed peripheral P188, and polyethylene glycol (PEG) infusion. Heart tissue was collected after 4 hours of reperfusion. Assessment of mitochondrial function or infarct size was performed. RESULTS: Mitochondrial yield improved significantly with IC P188 treatment compared to control animals (0.25% vs. 0.13%) suggesting improved mitochondrial morphology and survival. Mitochondrial respiration and calcium retention were also significantly improved with immediate IC P188 compared to controls (complex I RCI: 7.4 vs. 3.7 and calcium retention (nmol): 1152 vs. 386). This benefit was only observed with activation of complex I of the mitochondrial respiratory chain suggesting a specific impact of ischemia and reperfusion on this complex. Infarct size and serum troponin I were significantly reduced by immediate IC P188 infusion (infarct size: 13.9% vs. 41.1% and troponin I (µg/L): 19.2 vs. 77.4 µg/L). Delayed P188 and PEG infusion did not provide a significant benefit. CONCLUSIONS: Intracoronary infusion of P188 immediately upon reperfusion significantly reduces cellular and mitochondrial injury after ischemia and reperfusion in this clinically relevant porcine model of STEMI. The timing and route of delivery were critical to achieve the benefit.

Guidelines for the isolation and characterization of murine vascular smooth muscle cells. A report from the International Society of Cardiovascular Translational Research.

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.

Cardiac mitochondrial proteome dynamics with heavy water reveals stable rate of mitochondrial protein synthesis in heart failure despite decline in mitochondrial oxidative capacity.

We recently developed a method to measure mitochondrial proteome dynamics with heavy water ((2)H2O)-based metabolic labeling and high resolution mass spectrometry. We reported the half-lives and synthesis rates of several proteins in the two cardiac mitochondrial subpopulations, subsarcolemmal and interfibrillar (SSM and IFM), in Sprague Dawley rats. In the present study, we tested the hypothesis that the mitochondrial protein synthesis rate is reduced in heart failure, with possible differential changes in SSM versus IFM. Six to seven week old male Sprague Dawley rats underwent transverse aortic constriction (TAC) and developed moderate heart failure after 22weeks. Heart failure and sham rats of the same age received heavy water (5% in drinking water) for up to 80days. Cardiac SSM and IFM were isolated from both groups and the proteins were separated by 1D gel electrophoresis. Heart failure reduced protein content and increased the turnover rate of several proteins involved in fatty acid oxidation, electron transport chain and ATP synthesis, while it decreased the turnover of other proteins, including pyruvate dehydrogenase subunit in IFM, but not in SSM. Because of these bidirectional changes, the average overall half-life of proteins was not altered by heart failure in both SSM and IFM. The kinetic measurements of individual mitochondrial proteins presented in this study may contribute to a better understanding of the mechanisms responsible for mitochondrial alterations in the failing heart.

Effect of a high-protein diet on development of heart failure in response to pressure overload.

Heart failure treatment guidelines provide no recommendations regarding the intake of protein, though it has been proposed that increasing protein intake may result in clinical improvement. High-protein intake might improve protein synthesis and cell function, and prevent deterioration in mitochondrial and left ventricular function. We assessed the effects of a high-protein diet on the development of heart failure characterized by cardiac hypertrophy, impaired mitochondrial oxidative metabolism and contractile dysfunction induced by transverse aortic constriction in rats. A standard diet with 18% of energy intake from protein was compared with a high-protein diet (30% of energy intake). First, we evaluated the effects of protein intake on the development of heart failure during 14 weeks of aortic constriction, and found similar cardiac hypertrophy, contractile dysfunction, ventricular dilation, and decreased cardiac mitochondrial oxidative capacity with both 18% and 30% protein. We then assessed more advanced heart failure, with 22 weeks of aortic constriction. We again saw no difference in cardiac mass, left ventricular volume, mitochondrial oxidative capacity or resistance to permeability transition between the 18% and 30% protein diets. There was a modest but significant decrease in survival with heart failure with the 30% protein diet compared with 18% protein (p < 0.003). In conclusion, consumption of a high-protein diet did not affect cardiac mass, left ventricular volumes or ejection fraction, or myocardial mitochondrial oxidative capacity in rats with pressure overload induced heart failure, but significantly decreased survival.

Docosahexaenoic acid supplementation alters key properties of cardiac mitochondria and modestly attenuates development of left ventricular dysfunction in pressure overload-induced heart failure.

PURPOSE: Supplementation with the n3 polyunsaturated fatty acid docosahexaenoic acid (DHA) is beneficial in heart failure patients, however the mechanisms are unclear. DHA is incorporated into membrane phospholipids, which may prevent mitochondrial dysfunction. Thus we assessed the effects of DHA supplementation on cardiac mitochondria and the development of heart failure caused by aortic pressure overload. METHODS: Pathological cardiac hypertrophy was generated in rats by thoracic aortic constriction. Animals were fed either a standard diet or were supplemented with DHA (2.3 % of energy intake). RESULTS: After 14 weeks, heart failure was evident by left ventricular hypertrophy and chamber enlargement compared to shams. Left ventricle fractional shortening was unaffected by DHA treatment in sham animals (44.1 ± 1.6 % vs. 43.5 ± 2.2 % for standard diet and DHA, respectively), and decreased with heart failure in both treatment groups, but to a lesser extent in DHA treated animals (34.9 ± 1.7 %) than with the standard diet (29.7 ± 1.5 %, P < 0.03). DHA supplementation increased DHA content in mitochondrial phospholipids and decreased membrane viscosity. Myocardial mitochondrial oxidative capacity was decreased by heart failure and unaffected by DHA. DHA treatment enhanced Ca(2+) uptake by subsarcolemmal mitochondria in both sham and heart failure groups. Further, DHA lessened Ca(2+)-induced mitochondria swelling, an index of permeability transition, in heart failure animals. Heart failure increased hydrogen peroxide-induced mitochondrial permeability transition compared to sham, which was partially attenuated in interfibrillar mitochondria by treatment with DHA. CONCLUSIONS: DHA decreased mitochondrial membrane viscosity and accelerated Ca(2+) uptake, and attenuated susceptibility to mitochondrial permeability transition and development of left ventricular dysfunction.

Evaluation of docosahexaenoic acid in a dog model of hypertension induced left ventricular hypertrophy.

Marine n-3 polyunsaturated fatty acids alter cardiac phospholipids and prevent cardiac pathology in rodents subjected to pressure overload. This approach has not been evaluated in humans or large animals with hypertension-induced pathological hypertrophy. We evaluated docosahexaenoic acid (DHA) in old female dogs with hypertension caused by 16 weeks of aldosterone infusion. Aldosterone-induced hypertension resulted in concentric left ventricular (LV) hypertrophy and impaired diastolic function in placebo-treated dogs. DHA supplementation increased DHA and depleted arachidonic acid in cardiac phospholipids, but did not improve LV parameters compared to placebo. Surprisingly, DHA significantly increased serum aldosterone concentration and blood pressure compared to placebo. Cardiac mitochondrial yield was decreased in placebo-treated hypertensive dogs compared to normal animals, which was prevented by DHA. Extensive analysis of mitochondrial function found no differences between DHA and placebo groups. In conclusion, DHA did not favorably impact mitochondrial or LV function in aldosterone hypertensive dogs.

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria.

Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.

Glucose 6-phosphate dehydrogenase deficiency increases redox stress and moderately accelerates the development of heart failure.

BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) is the most common deficient enzyme in the world. In failing hearts, G6PD is upregulated and generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is used by the glutathione pathway to remove reactive oxygen species but also as a substrate by reactive oxygen species-generating enzymes. Therefore, G6PD deficiency might prevent heart failure by decreasing NADPH and reactive oxygen species production. METHODS AND RESULTS: This hypothesis was evaluated in a mouse model of human G6PD deficiency (G6PDX mice, ≈40% normal activity). Myocardial infarction with 3 months follow-up resulted in left ventricular dilation and dysfunction in both wild-type and G6PDX mice but significantly greater end diastolic volume and wall thinning in G6PDX mice. Similarly, pressure overload induced by transverse aortic constriction (TAC) for 6 weeks caused greater left ventricular dilation in G6PDX mice than wild-type mice. We further stressed transverse aortic constriction mice by feeding a high fructose diet to increase flux through G6PD and reactive oxygen species production and again observed worse left ventricular remodeling and a lower ejection fraction in G6PDX than wild-type mice. Tissue content of lipid peroxidation products was increased in G6PDX mice in response to infarction and aconitase activity was decreased with transverse aortic constriction, suggesting that G6PD deficiency increases myocardial oxidative stress and subsequent damage. CONCLUSIONS: Contrary to our hypothesis, G6PD deficiency increased redox stress in response to infarction or pressure overload. However, we found only a modest acceleration of left ventricular remodeling, suggesting that, in individuals with G6PD deficiency and concurrent hypertension or myocardial infarction, the risk for developing heart failure is higher but limited by compensatory mechanisms.

Enhanced resistance to permeability transition in interfibrillar cardiac mitochondria in dogs: effects of aging and long-term aldosterone infusion.

Functional differences between subsarcolemmal and interfibrillar cardiac mitochondria (SSM and IFM) have been observed with aging and pathological conditions in rodents. Results are contradictory, and there is little information from large animal models. We assessed the respiratory function and resistance to mitochondrial permeability transition (MPT) in SSM and IFM from healthy young (1 yr) and old (8 yr) female beagles and in old beagles with hypertension and left ventricular (LV) wall thickening induced by 16 wk of aldosterone infusion. MPT was assessed in SSM and IFM by Ca(2+) retention and swelling. Healthy young and old beagles had similar mitochondrial structure, respiratory function, and Ca(2+)-induced MPT within SSM and IFM subpopulations. On the other hand, oxidative capacity and resistance to Ca(2+)-induced MPT were significantly greater in IFM compared with SSM in all groups. Old beagles treated with aldosterone had greater LV wall thickness and worse diastolic filling but normal LV chamber volume and systolic function. Treatment with aldosterone did not alter mitochondrial respiratory function but accelerated Ca(2+)-induced MPT in SSM, but not IFM, compared with healthy old and young beagles. In conclusion, in a large animal model, oxidative capacity and resistance to MPT were greater in IFM than in SSM. Furthermore, aldosterone infusion increased susceptibility to MPT in SSM, but not IFM. Together this suggests that SSM are less resilient to acute stress than IFM in the healthy heart and are more susceptible to the development of pathology with chronic stress.

Effects of glucose-6-phosphate dehydrogenase deficiency on the metabolic and cardiac responses to obesogenic or high-fructose diets.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzymopathy that affects cellular redox status and may lower flux into nonoxidative pathways of glucose metabolism. Oxidative stress may worsen systemic glucose tolerance and cardiometabolic syndrome. We hypothesized that G6PD deficiency exacerbates diet-induced systemic metabolic dysfunction by increasing oxidative stress but in myocardium prevents diet-induced oxidative stress and pathology. WT and G6PD-deficient (G6PDX) mice received a standard high-starch diet, a high-fat/high-sucrose diet to induce obesity (DIO), or a high-fructose diet. After 31 wk, DIO increased adipose and body mass compared with the high-starch diet but to a greater extent in G6PDX than WT mice (24 and 20% lower, respectively). Serum free fatty acids were increased by 77% and triglycerides by 90% in G6PDX mice, but not in WT mice, by DIO and high-fructose intake. G6PD deficiency did not affect glucose tolerance or the increased insulin levels seen in WT mice. There was no diet-induced hypertension or cardiac dysfunction in either mouse strain. However, G6PD deficiency increased aconitase activity by 42% and blunted markers of nonoxidative glucose pathway activation in myocardium, including the hexosamine biosynthetic pathway activation and advanced glycation end product formation. These results reveal a complex interplay between diet-induced metabolic effects and G6PD deficiency, where G6PD deficiency decreases weight gain and hyperinsulinemia with DIO, but elevates serum free fatty acids, without affecting glucose tolerance. On the other hand, it modestly suppressed indexes of glucose flux into nonoxidative pathways in myocardium, suggesting potential protective effects.